The predominant mode of HIV transmission is heterosexual intercourse [1–3], during which the genital mucosa is the first tissue that comes in contact with HIV and innate and acquired mucosal immunity are the first barriers that are encountered by the virus [4–8]. Sexual transmission of HIV depends on the infectiousness of the HIV-seropositive patient and on the susceptibility of the uninfected partner [9,10]. It was recently reported that the probability of HIV transmission per coital act in an African cohort of HIV discordant sexual partners ranges between 0.001 (HIV viral load in the infected partner, < 1400 copies/ml) and 0.0023 (viral load, > 38 500 copies/ml) [11,12].
Susceptibility to HIV infection seems to vary widely among individuals. It is known that individuals can be identified who are repeatedly exposed to HIV in whom neither infection nor disease are seen (for reviews see [13,14]). Absence of HIV infection following exposure to HIV is associated, in a minority of exposed- seronegative (ESN) individuals, with mutations in the CCR5 co-receptor [15–18]. It has nevertheless become apparent that immune mechanisms have a pivotal role in this phenomenon [19,20] and numerous reports have documented the presence of HIV-specific cell-mediated immunity in ESN. Cell-mediated immune responses observed in ESN include HIV-specific CD4 and type 1 cytokine-secreting T-helper cells [21–23], CD4 β-chemokine-producing T-helper cells , and CD8 and MHC class I-restricted cytotoxic T-lymphocytes (CTL) [25–27]. HIV-specific cytotoxic T-cell activity has also been documented in an HIV exposed but uninfected infant . HIV-1-specific mucosal IgA antibodies may also be present in some but not all groups of ESN  and have been observed in cervical secretions of Italian heterosexual women  and of African [31–33] and Thai  commercial sex workers. IgA purified from these cervical secretions and saliva of ESN neutralize HIV-1  and inhibit HIV-1 transcytosis across a model of the mucosal epithelium [36,37].
Mucosal immune correlates of protection have been analysed in ESN women but no data is available on mucosal HIV-specific immunity in exposed but uninfected heterosexual men. To address this issue we selected a group of repeatedly, and recently HIV-exposed heterosexual male partners of HIV-seropositive women. Mucosal immunity was analysed in these individuals using seminal fluid and urethral swabs; HIV-systemic immunity was also analysed by the examination of peripheral blood mononuclear cells.
Materials and methods
Fourteen heterosexual couples discordant for HIV serostatus were enrolled in the study. In each couple the female partner was HIV-infected and the male partner was HIV-seronegative despite a prolonged history of penetrative sexual intercourse without condom use (and no other known risk factors). Inclusion criteria for the ESN was a history of multiple unprotected sexual episodes for at least 4 years with at least an episode of at-risk intercourse within the 4 months prior to the study period. Self-administered questionnaires show that the couples reported an average of 14 unprotected sexual episodes per year (range, 4 to > 40) in the 4 years before the study; vaginal sex was the norm and oral sex was practiced only very rarely (two couples engaged in vaginal and, rarely, orogenital sex). Anal sex was not reported by any couple. The epidemiological and clinical characterization of these couples is shown in Table 1.
Seven age-matched HIV-seropositive men [two Centers for Disease Control and Prevention (CDC) class A1, two CDC class A2 and three CDC class 3 B1] and seven age-matched healthy male volunteers without any known risk factor for HIV-1 infection were also enrolled in the study. All of the individuals enrolled in the study underwent careful medical evaluation that did not reveal concomitant infectious diseases. The heterosexual HIV discordant couples and the HIV-seropositive controls had been longitudinally followed for at least 3 years (prior to the study period) by the Department of Infectious Diseases of Santa Maria Annunziata Hospital in Florence, Italy. This allowed us to exclude from the study ESN in whom sexually transmitted diseases or any other infectious pathology had been reported during that time period. The presence of these pathologies in the anamnesis excluded some healthy controls from the study. A heterozygous CCR5-Δ32 deletion was detected in one of the 14 ESN. All analyses were performed in blinded manner.
The Research Ethics Committees of the Luigi Sacco Hospital, Milano and of the Santa Maria Annunziata Hospital, Florence, Italy, approved this protocol. Written informed consent was obtained from all patients before enrolment.
Sample collection and processing
Whole blood was collected by venopuncture in EDTA-containing vacutainer tubes (Becton Dickinson & Co., Rutherford, New Jersey, USA). Peripheral blood mononuclear cells (PBMC) were separated on lymphocyte separation medium (Organon Teknika Corp., Boxtel, the Netherlands), washed twice in phosphate-buffered saline and the number of viable leukocytes was determined by Trypan blue exclusion. To collect urethral samples, swabs were inserted in the urethra and gently rotated. Swabs were then introduced in a sterile Nalgene vial; the tip of the swab was pressed against the wall of the vial to allow release of the collected fluid from the swab and into the vial. A total of 500 μl material was collected (two swabs were performed when necessary) and diluted 1 : 4 in sterile saline. Ejaculates (obtained by masturbation) were collected in a sterile container. Seminal fluid mononuclear cells (SFMC) were separated, as described previously  by density gradient centrifugation (Percoll 47%, Percoll 90%; Sigma, St. Louis, Missouri, USA). Cells were washed twice in phosphate-buffered saline and the number of viable lymphocytes was determined by Trypan blue exclusion.
HIV RNA viral load was quantified using the AMPLICOR HIV Monitor test (Roche Diagnostics System, Nutley, New Jersey, USA) accordingly to the manufacturer's instructions. HIV complementary DNA (cDNA) detection was performed on seminal fluid. Five μl seminal fluid were centrifuged (1500 rpm, 15 min.) and DNA was extracted from 200 μg of pellet material by using the QIAamp Tissue Kit (Qiagen GmbH, Hilden, Germany). Fifty μl of the DNA extraction and 10 μl MgCl2 (2.5 mM) were then added to 50 μl amplification mix and processed according to the Roche protocol.
Measurement of HIV-1-specific antibodies
A modified Calypte Biomedical HIV-1 enzyme immunoassay (EIA) test (Calypte Biomedical Corporation, Berkeley, California, USA), based on a recombinant HIV-1 envelope protein, was used for determining the presence of IgG and IgA HIV-specific antibodies as follows. Samples with 25 μl sample buffer are added to the well and incubated at 37°C for 1 h. If antibodies to the HIV envelope proteins are present in the sample, they bind to the antigen immobilized to the well. The sample buffer significantly reduces the non-specific binding of antibodies and other proteins to the wells. A wash step removes the unbound material. Antibodies are detected using a modified EIA. In the first step, samples are incubated 1 h in the Calypte Biomedical HIV-1 coated microtiter strips with their buffer. After absorption of antibodies, a washing step is performed. A specific horseradish peroxidase-conjugated anti-human IgA, (Jackson ImmunoResearch Lab., West Grove, Pennsylvania, USA) is added to each well and incubated. After a second washing step, TMB, as chromogen and urea hydrogen peroxide, as substrate for peroxidase, are added. Stop solution containing 1 M H2SO4 blocks the reaction. The absorbance values are determined at 450 nm (A450),
Interferon (IFN)-γ secretion by antigen and mitogen-stimulated CD4 T lymphocytes
PBMC were resuspended in 200 μl RPMI1640 media (Sigma) supplemented with 2% AB serum (Sigma). PBMC were incubated for 18 h with: (i) a pool of previously described five synthetic peptides from the gp160 envelope of HIV-1 (final concentration 2.5 μM; Env) ; (ii) gag p17 (2 μg/ml; Austral Biological, San Ramon, California, USA); (iii) gag p24 (0.05 μg/ml; Protein Sciences Co., Meriden, Connecticut, USA); and (iv) influenza virus vaccine (A/Taiwan, A/Shanghai and B/Victoria; 24 μg/l; final dilution 1 : 1000; control; Flu). Antibody to CD28 (R&D Systems, Minneapolis, Minnesota, USA) was added during incubation at a dose of 1 μg/well to facilitate co-stimulation.
Cytometric analyses of intracytoplasmic lymphocyte cytokine producing cells were performed using an EPICS XL flow cytometer (Beckman-Coulter Inc., Miami, Florida, USA) equipped with a single 15-mW argon ion laser operating at 488 nm interfaced with 486 DX2 IBM computer (IBM, UK). For each analysis, 20 000 events were acquired and gated on CD4 or CD8 expression and side-scatter properties. Green fluorescence from fluorescein isothiocyanate (FL1) was collected through a 525 nm band-pass filter, orange–red fluorescence from R-phycoerythrin (FL2) was collected through a 575 nm band-pass filter and deep-red fluorescence from thiocyanate (TC) (FL4) was collected through a 670 nm band-pass filter. Data were collected using linear amplifiers for forward- and side-scatter and logarithmic amplifiers for FL1, FL2 and FL4. Samples were first run using isotype controls or single fluorochrome-stained preparations for colour compensation.
IFN-γ secretion by antigen and mitogen-stimulated CD8 lymphocytes: ELIspot assayes
To purify CD8 T lymphocytes, PBMC were first incubated for 1 h in RPMI containing 20% AB serum in plastic flasks to remove monocytes and dendritic cells. Monocyte-depleted cells were then incubated with 20 μl MACS CD8 Microbeads (Miltenyi Biotec, Auburn, California, USA) for 15 min at 4°C and magnetic separation was performed using a miniMACS separator. Purity was defined by FACS analysis of positive and negative fractions, labelling cells with CD8, CD4 and CD14 monoclonal antibodies (mAb) and was always > 99%. Plastic adherent cells (monocytes and dendritic cells) were removed from the flask using a scraper and were incubated (3 h, 37°C, 7% CO2) in duplicate in 96-well nitrocellulose plates coated with IFNγ mAb (MABTECH, Nacka, Sweden). Cells were either unstimulated (medium), or stimulated with a pool of five synthetic peptides from gp160 of HIV-1 (20 μM final concentration; Env), gag p17; gag p24; or Flu (control antigen). After 3 h purified CD8 lymphocytes were added to the plastic adherent cells culture at a ratio of 10 : 1. The five peptides used in the stimulation (Env T1; Env T2; Env Th4; Env P18IIIB; Env p18MN) were predicted to be immunogenic because of amphipathic helical folding potential  and are promiscuous as they are recognized by multiple HLA Class I molecules (including HLA A1, A2 and B8) . Assays used 2 × 105 cells/well. Plates were incubated overnight at 37°C in 7% CO2, then the cells were discarded and the plates incubated at room temperature for 3 h. A second biotinylated anti IFNγ mAb (7-B6-1 biotin; MABTECH), followed by streptavidin-conjugated alkaline phosphatase (MABTECH) for 2 h, was subsequently used. Individual IFNγ -producing cells were detected as dark blue spots using an alkaline phospatase conjugate substrate kit (Bio-Rad laboratories, Hercules, California, USA). Spots were counted by visualization with a dissecting microscope (× 40). HIV-1-specific responses were reported as number of spot-forming units (SFU)/ × 106 mononuclear cells after subtraction of background IFNγ secretion. A positive response was scored if HIV-1-stimulated SFU exceeded background by a factor of > 2.
Lymphocyte subsets were evaluated in both PBMC and SFMC using an Epics XL flow-cytometer (Coulter Electronics Inc., Miami Lakes, Florida, USA) using 50 μl of EDTA-treated peripheral blood or 5 × 105 SFMC incubated for 30 min at 4°C with fluorochrome labelled mAb. Erytrocyte lysis was obtained after incubation with the Immuno-Prep Epics Kit (Coulter Electronics) and Q-prep Work Station (Coulter Electronics). Lymphocytes were selectively analysed using forward- and side-scatter properties (FSC, SSC). For each sample, multiparametric data were acquired for 5000 events.
The statistical analysis was based on a non-parametric Jonckheere–Terpstra test for trends. All data were also analysed by a non-parametric Kruskal–Wallis test. Comparisons between the different groups were made using a two-tailed t test. Possible relationships were evaluated using a Pearson's correlation test. Statistical analysis was performed using the SPSS statistical package (SPSS Inc. Chicago, Illinois, USA)
Clinical and epidemiological characterization of the individuals enrolled in the protocol
Plasma HIV-1 RNA was quantified in the HIV-seropositive partners to verify whether absence of infection could be seen even in the presence of a significant HIV-1 viral load in the infected partner. A spectrum of values ranging from < 400 to 28 500 RNA copies/ml was observed in the infected female partners. Thus, no threshold correlated with a more likely sexual transmission of HIV-1 in these exposed individuals (Table 1). HIV-1 plasma viraemia was undetectable in all of the ESN men. To verify the possibility of a mucosally-confined presence of HIV-1 in the ESN men, cDNA in seminal fluid was analysed. HIV-1 cDNA was detected in all of the HIV-1-infected controls but in neither the ESN nor in the low-risk controls (data not shown).
HIV-1-specific IgG and IgA in urethral swabs of ESN, HIV-seropositive patients and controls
Urethral swabs were examined for the presence of HIV-specific IgA and IgG. A positive titre of urethral HIV-1-specific IgA [optical density (OD), > 2 SD above the mean OD observed in low-risk controls, 0.232] was detected in 11 out of 14 (78%) ESN, six out of seven (71%) HIV-seropositive patients and none of seven (0%) low-risk controls. The mean OD were: ESN, 0.488; HIV-seropositive individuals, 0.501; low-risk controls, 0.172 (ESN versus HIV-seropositive individuals, not significant; ESN versus low-risk controls, P = 0.002; HIV-seropositive individuals versus low-risk controls, P = 0.03; Fig. 1a).
In contrast, urethral HIV-specific IgG (OD, > 2 SD above the mean OD observed in low-risk controls, 0.012) were detected in all HIV-seropositive individuals, but in neither ESN nor low-risk controls. The mean OD were: ESN, 0.0076; HIV-seropositive individuals, 0.38; low-risk controls, 0.0073. (ESN versus HIV-seropositive individuals, P = 0.001; ESN versus low-risk controls, non-significant; HIV-seropositive individuals versus low-risk controls: P = 0.001; Fig. 1b).
Env-, gag p17-, and gag p24-stimulated IFNγ-secretion by CD4 lymphocytes
PBMC of all the individuals enrolled in the protocol were stimulated in vitro with a pool of five different synthetic HIV-1 Env peptides, Gag p17, Gag p24, or Flu (non-HIV control antigen) Results showed that the frequency of Env-, p17- and p24-IFNγ-producing CD4 T cells was not statistically different between ESN individuals and HIV-seropositive patients, even if the highest frequency of IFNγ-producing, HIV-specific CD4 T cells for all three antigens was observed in ESN individuals. In more detail, the mean frequency of: Env-specific, IFNγ-secreting lymphocytes was increased in ESN compared with low-risk controls (P = 0.04); p17-specific, IFNγ-secreting lymphocytes was augmented in ESN and HIV-seropositive patients compared with low-risk controls (P = 0.04 and 0.005, respectively); and p24-specific, IFNγ-secreting lymphocytes was higher in ESN and HIV-seropositive patients compared with low-risk controls (P = 0.02 and 0.04, respectively) (Fig. 2). Finally, the frequency of Flu-stimulated, IFNγ-producing CD4 T cells (control antigen) was comparable in ESN and low-risk controls (0.09 and 0.083, respectively), but was lower in HIV-seropositive patients (0.036).
Env- Gag p17- and Gag p24-stimulated IFNγ secretion by CD8 lymphocytes
Env-, Gag p17- and Gag p24-stimulated IFNγ production by purified CD8 T cells was analysed in nine ESN, six HIV-seropositive patients and six low-risk controls by using an ELIspot assay. Flu was used as a non-HIV antigen control. Results showed that the number of HIV-specific CD8 IFNγ-secreting cells was similar in ESN and HIV-seropositive patients. In this case, the most robust responses were detected in HIV-seropositive patients. Thus: Env-specific and IFNγ-producing CD8 T cells were augmented in ESN and HIV-seropositive patients compared with low-risk controls (P = 0.04 and 0.03, respectively); p17-specific CD8 T cells were increased in ESN and HIV-seropositive patients compared with low-risk controls (P = 0.01 and 0.001, respectively); and CD8 T cells producing IFNγ in response to p24 were more frequent in ESN and HIV subjects compared with low-risk controls (P = 0.01 and 0.008, respectively). The number of Flu-specific CD8 and IFNγ-producing lymphocytes was similar in ESN and low-risk controls, but was significantly reduced in HIV-seropositive patients (P = 0.02; Fig. 3).
Immunophenotypic analyses on PBMC and SFMC
The expression of a panel of immunophenotypic marker was evaluated in PBMC and in SFMC. Significant results observed upon analysis of PBMC can be summarized as follows: CD4 T lymphocytes were reduced in ESN compared with low-risk controls (P = 0.03); CD8 T lymphocytes were increased in ESN compared with low-risk controls (P = 0.02); and CD4 naive lymphocytes were reduced (P = 0.02) and CD4 memory cells were increased in ESN compared to low-risk controls (data not shown).
It is of note that whereas the percentage of activated CD4 and CD8 T lymphocytes was similar in PBMC of ESN and healthy controls, both CD4CD25 and CD8CD38RO T cells were significantly augmented in SFMC of ESN compared with SFMC of healthy controls (P = 0.005 and 0.04, respectively; Fig. 4). The percentage of CD4CD25 and CD8CD38RO cells in the seminal fluid was statistically correlated (r, 0.890; P = 0.003).
Correlations between immune parameters in the ESN and viro-immunologic data of the HIV-seropositive partners
p17-specific, IFNγ-producing CD8 T lymphocytes correlated with the percentage of CD4 T cells observed in the HIV partner (r, 0.67; P = 0.031). Interestingly, the urethral concentration of HIV-specific IgA tended to be higher in those ESN who reported having at-risk sexual episode closest to the time of collection (r, −0.53; P = 0.049; see Table 1).
Mucosal and systemic immune correlates of protection against HIV infection were analysed in 14 heterosexual couples in whom the male partner was HIV-1- uninfected despite a prolonged history of penetrative sexual intercourse without condom use with an HIV-seropositive partner. The male ESN partners were carefully selected based on the date of the last at-risk sexual episode (within 4 months prior to the study) and on the absence of any concomitant diseases. Results showed that unprotected sexual activity not resulting in apparent HIV infection is associated, in ESN men, with the activation of HIV-1-specific mucosal and systemic immunity. Thus, mucosal HIV-1-specific IgA and systemic HIV-1-specific CD4 IFNγ-producing cells as well as HIV-1-specific CD8 T lymphocytes were present in ESN men. Results also showed that immune activation is detected in lymphocytes purified from seminal fluid but not in peripheral blood lymphocytes. These findings confirm and extend previous studies in which HIV sexually-exposed ESN women were analysed [30–37,42] and, for the first time, report the presence of HIV-1-specific mucosal immunity in men.
It is interesting that whereas increased amounts of activation marker-bearing immune cells were detected both in vaginal washes and in peripheral blood of ESN women , activated lymphocytes are seen in seminal fluid but not in the peripheral blood of ESN men. Analogously, serum HIV-specific IgA was seen in ESN women  but not detected in ESN men (data not shown). These data suggest that the anatomical barriers characteristic of the male genital tract are more impenetrable to viruses than the barriers of the female genital region. Alternatively, mucosal immunity could be better at containing limited HIV infection in males than in females.
The observation that HIV-specific CD4 T cells are observed in the peripheral blood of ESN men demonstrates that lymphocyte priming by HIV or by viral antigens occurs in ESN. The detection of HIV-specific, IFNγ-secreting CD8 T lymphocytes in ESN men could suggest that sexual exposure to HIV is associated with an actual abortive infection and that live, replicating virus could have penetrated the mucosal barriers and stimulated systemic specific immunity. In fact, only actual infection with the virus would result in presentation of viral antigens in association with HLA class I molecules and elicitation of a CD8-mediated immune response (reviewed in ). Gag is a structural HIV protein that is translated from primary HIV transcripts (reviewed in [6,44]). Thus, the observation that Gag-specific CTL are detected in ESN further reinforces the idea that live replication-competent virus might have been encountered and controlled by immune system of these individuals. This possibility notwithstanding, it is of note that HIV-specific, IFNγ-secreting CD8 T lymphocytes were detected despite the fact that Gag proteins were used in the CTL assays. Thus, even if the conventional presentation of peptides within HLA class I molecules classically involves processing of protein antigens expressed within the host cells, it is has been recently shown that exogenous antigens can also be processed for presentation by MHC class I molecules via alternate antigen/processing mechanisms [45–47]. Interestingly, this alternate processing/presentation pathway is suggested to be important in HIV infection . Thus, the detection of HIV-specific CTL in ESN individuals might not be a consequence of abortive infection, but rather could result from alternate processing mechanisms by dendritic cells. This second explanation is apparently reinforced by the observation that HIV cDNA was not detected in blood or seminal fluid of the ESN men. Further in-depth analyses will be necessary to answer this still unresolved question.
The observation that the urethral concentration of HIV-1-specific IgA tended to be higher in those ESN who reported having at-risk sexual episodes closest to the time of collection could be explained in at least two ways. Firstly, high IgA levels could be due, at least in part, to the persistence of cervico-vaginal IgA of the infected partners. More intriguing is the possibility that repeated exposure to HIV might be necessary to maintain protective immunity. This hypothesis is supported by previous data showing that (i) HIV-specific T helper and CTL responses disappear within 6–9 months after cessation of exposure to the virus in the uninfected newborns of HIV-seropositive women [21,49] and in health care workers reporting a single professional exposure to HIV-seropositivebody fluids ; (ii) the concentration of HIV-specific plasma IgA is significantly diminished in ESN women who adopt safe sex procedures ; and (iii) late seroconversion occurring in Kenyan HIV-resistant sex workers who interrupt commercial sex work is associated with the waning of HIV-specific CD8 responses due to reduced antigenic exposure . Therefore, the maintenance of possibly protective HIV-specific cell-mediated and humoral immunity might be contingent upon repeated specific immune stimulation.
Our working hypothesis was that the presence of HIV-specific seminal IgA would be responsible for neutralization of HIV infectivity. It is known that IgA can inhibit transcytosis of HIV across epithelial barriers [36,51]. Nevertheless a number of factors make this hypothesis particularly complex. The quality of the HIV-specific IgA present in ESN might not be uniform in ESN, and IgA with the ability to recognize different HIV epitopes and with diverse neutralizing ability could be important following exposure to the virus. Alternatively, neutralization of HIV infectivity might be mediated by factors other than IgA. The first possibility is supported by data showing that the epitope specificity of plasma IgA detected in ESN and in HIV-infected women is indeed different  and by a recent report showing that CD8 T lymphocytes respond to different HIV-1 epitopes in seronegative and infected subjects . The second hypothesis is indirectly supported by the observation that CD8 T cells are augmented in the peripheral blood of ESN men. These lymphocytes are known to produce an array of antiviral factors, including CAF [54,55] and β-chemokines (reviewed in ) that could decisively contribute to the suppression of HIV infectivition via seminal fluid. The concentration of beta chemokines in seminal fluid of ESN, HIV-seropositive patients and low-risk healthy controls was similar (data not shown). Because of limitations in the amount of available material, seminal and/or urethral CD8 T cells could not be characterized in this aspect; this possibility therefore requires further investigation.
The increase in peripheral CD8 T lymphocytes seen in ESN individuals is not paralleled by a similar increase in CD4 T cells, as this latter subset is instead reduced in ESN. Furthermore, a reduction in naive T lymphocytes and an increase in memory cells, with a resulting skewing of the memory : naive cell ratio, are detected in ESN. This result confirms previous data from analyses performed in ESN women  and in uninfected newborns of HIV-seropositive mothers , parallels the alterations seen in HIV-seropositive individuals and suggests that the same immunologic stimuli/signal that leads to phenotypic immune alterations in HIV-seropositive patients also occurs in ESN.
In conclusion, we analysed HIV-specific immune responses in heterosexual HIV-exposed ESN men and verified that cellular and humoral immune mechanisms are activated in these individuals. This is the first report of HIV-specific mucosal immunity in ESN men. These data add to the body of knowledge of the immune correlates present in exposed, uninfected individuals and could be important in vaccine design.
We thank H. B. Urnovitz and T. Gottfried, Calypte Biomedical, for providing us with material and useful insights.
Sponsorship: Supported the Istituto Superiore di Sanita III Programma Nazionale di Ricerca sull AIDS 1999, grants 40C/1.23; 30C.17 and 40C.292
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